Factor XIII, also known as fibrin stabilizing factor, fibrinoligase, or plasma transglutaminase, is a plasma glycoprotein that circulates in blood as a zymogen (Mr of about 320 kDa) complexed with fibrinogen. (Greenberg and Shuman, J. Biol. Chem. 257:6096-6101 (1982)). Plasma factor XIII zymogen is a tetramer consisting of two a subunits and two b subunits. Each subunit has a molecular weight of about 83,000 Da, and the complete protein has a molecular weight of approximately 320 kDa. The a subunit contains the catalytic site of the enzyme, while the b subunit is thought to stabilize the a subunit or to regulate the activation of factor XIII. The amino acid sequences of the a and b subunits are known. (Ichinose et al., Biochemistry 25:6900-6906 (1986); Ichinose et al., Biochemistry 25:4633-4638 (1986)). Recombinant expression of biologically active factor XIII has been described, see for example, EP 0 268 772 B1.
In vivo, activated factor XIII catalyzes cross-linking reactions between other protein molecules. During the final stages of blood coagulation, thrombin converts factor XIII zymogen to an intermediate form, which then dissociates in the presence of calcium ions to produce activated factor XIII. Placental factor XIII is activated upon cleavage by thrombin. Activated factor XIII is a transglutaminase that catalyzes the cross-linking of fibrin polymers through the formation of intermolecular ξ(δ)-glutamyl lysine bonds, thereby increasing clot strength. This cross-linking reaction requires the presence of calcium ions. Activated factor XIII also catalyzes the cross-linking of the δ-chain of fibrin to α2-plasmin inhibitor and fibronectin, as well as the cross-linking of collagen and fibronectin, which may be related to wound healing. The covalent incorporation of α2-plasmin inhibitor into the fibrin network can increase the resistance of the clot to lysis.
Factor XIII deficiency results in “delayed bleeding,” but does not affect primary hemostasis. Current treatment practices for patients having factor XIII deficiencies generally involve re-placement therapy with purified factor XIII isolated from natural sources.
Factor XIII is also useful in the treatment of patients with scleroderma (Delbarre et al., Lancet 2:204 (1984); Guillevin et al., Pharmatherapeutica 4:76-80 (1985); Grivaux and Pieron, Rev. Pnemnol. Clin. 43:102-103 (1987)), ulcerative colitis (Suzuki and Takamura, Throm. Haemostas. 58:509 (1987); U.S. Pat. No. 5,378,687), colitis pseudomembranous (Kuratsuji et al., Haemostasis 11:229-234 (1982) and as a prophylactic of rebleeding in patients with subarachnoid hemorrhage (Henze et al., Thromb. Haemostas. 58:513 (1987), in addition to other conditions. Furthermore, factor XIII has been demonstrated to be useful as a component of tissue adhesives (U.S. Pat. Nos. 4,414,976; 4,453,939; 4,377,572; 4,362,567; 4,298,598; 4,265,233 and U.K. Patent No. 2 102 811 B). Another use of factor XIII includes reducing blood loss in patients undergoing surgery (U.S. Pat. No. 5,607,917).
Methods for the recombinant production of human factor XIII are known in the art. See for example, European Patent No. 0 268 772 B1. Also, methods for the purification of factor XIII from biological sources using crystallization and/or precipitation steps combined with certain chromatography methods are known (U.S. Pat. Nos. 4,597,899; 5,204,447 and 5,612,456). Although each of the prior methods results in the enrichment of factor XIII, each of the methods is either complex and/or expensive for the isolation of factor XIII at high yield. What is needed in the art is a simpler, less expensive method for the isolation of highly purified factor XIII, i.e., compositions that are greater than 95% pure with respect to contaminating proteins would be particularly desired. Methods that can provide compositions comprising factor XIII that is 1% or less activated factor XIII, contain less than 2% protein aggregates, and/or less than 5% charge isomers of factor XIII, while providing a high yield would also be particularly desired.